Difference between revisions of "Team:KU Leuven/Research/Methods"
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<p><b>Theory</b><br/> | <p><b>Theory</b><br/> | ||
− | To be able to create patterns, two different cell types called A and B will interact with each other. In order to achieve the desired behavior, the cells used in the experiments were derived from K12 <i>Escherichia coli</i> strains with introduction of specific knockouts. Cell type A has a deletion of tar and tsr, whereas in cell type B both tar and cheZ are knocked out. The Keio collection is composed of a set of precisely defined single-gene deletions of all nonessential genes in E. coli K-12. The targeted genes were replaced by a kanamycin resistance cassette. The kanamycin cassette is enclosed between two FRT sites making excision possible using FLP recombinase (reference 1). FLP recombinase triggers an intramolecular recombination between FRT repeats in the chromosome. Since both the antibiotic resistance gene and the plasmid replication region are surrounded by two FRT sites both are to be eliminated (Figure 1, step 1). <br/> | + | To be able to create patterns, two different cell types called A and B will interact with each other. In order to achieve the desired behavior, the cells used in the experiments were derived from K12 <i>Escherichia coli</i> strains with introduction of specific knockouts. Cell type A has a deletion of <i>tar</i> and <i>tsr</i>, whereas in cell type B both <i>tar</i> and <i>cheZ</i> are knocked out. The Keio collection is composed of a set of precisely defined single-gene deletions of all nonessential genes in <i>E. coli</i> K-12. The targeted genes were replaced by a kanamycin resistance cassette. The kanamycin cassette is enclosed between two FRT sites making excision possible using FLP recombinase (reference 1). FLP recombinase triggers an intramolecular recombination between FRT repeats in the chromosome. Since both the antibiotic resistance gene and the plasmid replication region are surrounded by two FRT sites both are to be eliminated (Figure 1, step 1). <br/> |
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A genetic procedure for moving selectable mutations of interest called the P1 transduction was used. Since the packaging of the bacteriophage P1 is rather inaccurate, it will on occasion package the DNA of its bacterial host instead of its own phage chromosome. This implies that the lysate contains either packaged phage or bacterial DNA. After infection of a second host with this lysate, a transfer of parts of the chromosome from the donor strain into the receiver strain will take place. Those DNA pieces can then recombine using the FRT sites and hereby be incorporated permanently into the chromosome of the new strain. Here, the recombination was triggered by selection on kanamycin. (reference 2) <br/> | A genetic procedure for moving selectable mutations of interest called the P1 transduction was used. Since the packaging of the bacteriophage P1 is rather inaccurate, it will on occasion package the DNA of its bacterial host instead of its own phage chromosome. This implies that the lysate contains either packaged phage or bacterial DNA. After infection of a second host with this lysate, a transfer of parts of the chromosome from the donor strain into the receiver strain will take place. Those DNA pieces can then recombine using the FRT sites and hereby be incorporated permanently into the chromosome of the new strain. Here, the recombination was triggered by selection on kanamycin. (reference 2) <br/> | ||
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− | In general, we used three steps to obtain our double knock-outs (Figure 1). In the first step, the kanamycin cassette of our tar knock-out strain was removed by flippase, coded on plasmid PCP20. Afterwards, the temperature sensitive plasmid was removed by growing the cells overnight at 42°C. In a third step, the tar knock-out strain is infected by lysate originating from our tsr and cheZ knock-out strains. After selection on kanamycin plates, we obtained our double knock-outs. These knock-outs were confirmed by PCR. For more information, please check our result page. <br/> | + | In general, we used three steps to obtain our double knock-outs (Figure 1). In the first step, the kanamycin cassette of our <i>tar</i> knock-out strain was removed by flippase, coded on plasmid PCP20. Afterwards, the temperature sensitive plasmid was removed by growing the cells overnight at 42°C. In a third step, the <i>tar</i> knock-out strain is infected by lysate originating from our <i>tsr</i> and <i>cheZ</i> knock-out strains. After selection on kanamycin plates, we obtained our double knock-outs. These knock-outs were confirmed by PCR. For more information, please check our result page. <br/> |
Revision as of 12:17, 16 September 2015
Methods
On this page you can find all of the methods and protocols used in the lab to obtain our results. For some techniques, we included some basic theory, since it is a prerequisite to get acquainted with the theory behind these techniques before using them. To learn more about them, click the titles below!
Contact
Address: Celestijnenlaan 200G room 00.08 - 3001 Heverlee
Telephone: +32(0)16 32 73 19
Email: igem@chem.kuleuven.be